1. Field of the Invention
The invention relates to screening methods for one-bead-one-compound combinatorial libraries and includes a screening assay that uses live cells to identify synthetic ligands that promote attachment and growth or proliferation of epithelial and non-epithelial cells. Also included are ligands specific for epithelial and non-epithelial cancer cells.
2. Description of Related Art
One-bead-one-compound combinatorial bead libraries (see Lam, Kit S. et al. “A new type of synthetic peptide library for identifying ligand-binding activity.” Nature 354 (1991): 82-84), such as one-bead-one-compound peptide libraries, are being used to study cell adhesion properties of cancer cells. Using random peptide bead libraries and suspended cancer cells, peptide ligands that promote cell attachment have been identified for lymphoma (Park, Steven, Renil Manat, Brian Vikstrom, Nail Amro, and Kit S. Lam. “Identification of peptide ligands for α4β1 integrin receptor as potential targeting agents for non-Hodgkin's lymphoma,” abstract in Peptides: The Wave of the Future, 2nd International Peptide Symposium in conjunction with the 17th American Peptide Symposium, San Diego, Calif. (Jun. 9-14, 2001)) and prostate cancer cell lines (Pennington, Michael E., Kit S. Lam and Anne E. Cress. “The use of a combinatorial library method to isolate human tumor cell adhesion peptides.” Molecular Diversity 2 (1996): 19-28; DeRoock, Ian B., Michael E. Pennington, Thomas C. Sroka, Kit S. Lam, G. Tim Bowden, Elisabeth L. Bair, and Anne E. Cress. “Synthetic Peptides Inhibit Adhesion of Human Tumor Cells to Extracellular Matrix Proteins.” Cancer Research 61 (Apr. 15, 2001): 3308-13).
In the existing methods, live cells in suspension are incubated for about one to four hours with a bead library, and the library is then screened for beads with peptide ligands that promote cell attachment. This is done by visual selection the beads are examined under a dissecting microscope and those beads with attached cells are removed using a micropipet. Further steps are then performed to confirm that the removed beads are in fact capable of binding the particular type of cells tested. Then, the peptides on those beads are sequenced. (See Pennington et al., “Use of a combinatorial library method,” 19-28.)
In another existing method of testing live cells for peptide ligands that affect cell growth on culture plates, a bead library is prepared having selectively cleavable peptides such that a proportion of the peptide on each bead is attached to the bead by a cleavable linker. When the library is treated with a cleaving agent, enough of the peptides are released from the beads to cause the biological effect, and the rest of the peptides remain bound to the beads to allow for later sequencing. Suspended cells are incubated in tissue culture wells with a few beads and with peptides released from the beads. The effect of the released peptides on the cells (inhibition or stimulation of cell growth) is determined, and the corresponding beads are removed. The sequences of the attached peptides are then determined. (See U.S. Pat. No. 5,510,240, issued Apr. 23, 1996 to Lam, Kit S. et al.)
The existing methods, however, are not satisfactory in certain cases. The methods are difficult to use with epithelial cells, which include the majority of solid cancer cell cultures, such as lung cancer cells, that exist as adherent cultures rather than as suspended cells. With incubation periods of only a few hours, these cells are often only weakly attached to the beads and may easily fall off, rendering the screening method less accurate because some beads with attached cells are missed. Also, the existing methods may not detect cell surface receptors that may be altered by trypsin and/or EDTA. Trypsinization is commonly used to separate tissues or cell cultures into a single-cell suspension for testing with a combinatorial library. The treatment with trypsin may eliminate some, or alter the conformation of, cell surface receptors. In addition, the existing methods do not select for ligands that promote cell growth or proliferation, but, rather, for ligands involved in cell attachment, particularly short-term attachment.
Thus, there is a need for a screening assay that is specific and sensitive, works well with epithelial cells, can be used to detect cell surface receptors susceptible to trypsin, and selects for ligands that promote not only cell attachment, but also cell growth or proliferation.